"PROCESS FOR PRODUCING HETEROLOGOUS POLYPEPTIDES"

Abstract

A process is described for producing a polypeptide heterologous to E. coli wherein E. coli cells comprising nucleic acid encoding the polypeptide are cultured in a culture medium while feeding to the culture medium a transportable organophosphate, such that the nucleic acid is expressed. The polypeptide is then recovered from the cells.

Full Text

BACKGROUND OF THE INVENTION
Related Applications
This application claims priority to U.S. Provisional Application No.: 60/552,678
filed March 11,2004, to which U.S. Provisional Application this application claims
priority under 35 U.S.C. §119, the contents of which are incorporated herein by reference.
1. Field of die Invention
The invention relates to a process for producing a polypeptide heterologous to E.
coll. More particularly, the invention is directed to using organophosphate to improve
yield of such polypeptides.
2. Description of Related Art
Expression of heterologous proteins by Escherichia coli, aided by the wellunderstood
molecular biology and relative ease in genetic manipulation of the
microorganism, has been very productive in both laboratory and industry. Typically, an
inducible promoter (for example, the alkaline phosphatase promoter, the tac promoter, the
arabinose promoter, etc.) is employed for the regulation of heterologous protein
expression. The requirement of an induction event provides the researcher the
opportunity to manage the timing of expression of the target protein. This ability is
especially important for those heterologous proteins that are not well tolerated at high
concentrations by the host. By achieving desirable cell density prior to the induction of
expression, the volumetric yield of the desired protein may be maximized.
Cells cease to grow when the microorganism is deprived of a required nutrient.
The limiting component may be carbon, nitrogen, phosphate, oxygen or any of the
elements required by the cell. Under such conditions, the cells exit from the growth phase.
A way to alleviate the culture of the stress responses caused by the nutrient limitation is to
provide a feed of the lacking component. Common feeds introduced into fed-batch
fermentation processes include glucose, amino acids, oxygen, etc.
In the case of cellular phosphorus (P), the requirement for phosphate supply is not
surprising given that P is the fifth most abundant element in a cell behind carbon, oxygen,
nitrogen, and hydrogen. Slanier, Adelberg and Ingraham, The Microbial World. 4th ed.
(Prentice Hall, NJ 1976), p. 1357. Phosphorus is an essential component in numerous
macromolecules such as nucleic acids, liposaccharides and membrane lipids. Furthermore,
its role in the high-energy phosphoanhydride bonds makes it especially important in energy
metabolism. E. coli is capable of utilizing inorganic phosphate (Pi), organophosphate or
phosphonate as the primary P source. The uptake of Pi from the environment can be
achieved through two transporter systems, the Pit and the Pst systems. For the
organophosphates, most are non-transportable and they first need to be hydrolyzed
enzymatically in the periplasm before the released Pi can be taken up by the Pi transport
system(s). Only a few organophosphates are transportable, and glycerol-3-phosphate
(G3P) is one such example. G3P and glycerophosphate-1-phosphate (G1P) are known as
alpha-glycerophosphates. In response to Pi-limitation and carbon-limitation, E. coli is
capable of taking up available intact G3P from the external environment into the
intracellular compartment, where G3P is metabolized to yield needed phosphate or carbon.
Wanner, "Phosphorus Assimulation and Control of the Phosphate Regulon", in Escherichia
coli and Salmonella Cellular and Molecular Biology, Neidhardt, ed., (second edition),
American Society for Microbiology Press (1996), pp. 1357-1365.
Further references on G3P are Silhavy et al., J. Bacteriol. 126: 951-958 (1976) on
the periplasmic protein related to the sn-glycerol-3-phosphate transport system of E. coli;
Argast et al, J. Bacteriol. 136: 1070-1083 (1978) on a second transport system for snglycerol-
3-phosphate in E. coli; Elvin et al., J. Bacteriol. 161: 1054-1058 (1985) on Pi
exchange mediated by the g/pr-dependent G3P transport system; Rao et al., J. Bacteriol.
175: 74-79 (1993) on the effect ofglpT and glpD mutations on expression of \lnephoA gene
in E. coli; and Elashvili et al, Appl. Environ. Microbiol. 64: 2601-2608 (1998) onphnE
and glpT genes enhancing utilization of organophosphates in E. coli K-l 2. Further,
Vergeles et al, Eur. J. Biochem.. 233: 442-447 (1995) disclose the high efficiency of
glycerol-2-phosphate (G2P), otherwise known as beta-glycerophosphate, and G3P as
nucleotidyl acceptors in snake venom phosphodiesterase esterifications.
The current understanding of the two transport systems for the uptake of
exogenous G3P in E. coli, the Ugp and GlpT transport systems, has been well
summarized in the book Escherichia coli and Salmonella, Cellular and Molecular Biology
edited by Neidhardt et. al. (second edition), supra, pp. 1364 referring to references 13 and
81. The Ugp operon belongs to ihepho regulon. It is induced by phosphate limitation
and positively regulated byphoB protein. The Ugp system is a periplasmic binding
protein-dependent multi-component transport system, with ugpB encoding the
periplasmic binding protein, ugpA and ugpC encoding integral membrane channel
proteins, and ugpC encoding ATPase. GlpT is part of the glp system that mediates the
uptake and metabolism of glycerol, G3P, and glycerol phosphoryl phosphodiesters (Lin et
al, Annu. Rev. Microbiol. 30- 535-578 (1976); Chapter 20; pg 307-342 Dissimilatory
Pathways for sugars, polyols and carboxylates. Escherichia coli and Salmonella, Cellular
and Molecular Biology, second edition). This transport system is an anion exchanger that
is known to mediate the efflux of Pi from the cytoplasm by exchange with external G3P.
In a wild-type strain growing on G3P, while little Pi is released by cells taking up G3P
via the Ugp system, Pi can be released into the periplasm when G3P is taken up via the
GlpT system. If a repressive amount of Pi is released as a result of g/pr-permeasemediated
efflux, the to regulon activity, the Ugp system included, will be shut off.
Under certain conditions, GlpT is the only route for the exit of Pi from the cell by
exchange with external G3P. Elvin et al, J. Bacteriol. 161. 1054-1058 (1985);
Rosenberg, "Phosphate transport in prokaryotes," p. 205-248. In B. P. Rosen and S.
Silver (ed.), Ion Transport in Prokaryotes (Academic Press, Inc., New York, 1987).
When the capacities of the Ugp and the GlpT systems are compared to transport
G3P, the maximal velocities of the two systems are similar. The apparent affinity for
G3P is higher with the Ugp system than with the GlpT system. Likely, both systems will
be able to supply enough G3P for cell growth if available in the growth medium.
However, G3P transported exclusively via the Ugp system can serve as the sole source
only of phosphate but not of carbon, while GlpT-transported G3P can serve as the sole
source for both (Schweizer et al, J Bacteriol. ISO: 1154-1163 (1982)). The two ugp
genes coding for the 2o-regulon-dependent G3P transport system have been mapped
(Schweizer et al, J. Bacteriol. 150: 1164-1171 (1982)), the ugp region containing these
genes has been characterized (Schweizer et al, Mol and Gen. Genetics. 197: 161-168
(1984)), and the regulation of ugp operon studied (Schweizer et al., J. Bacteriol, 163:
392-394 (1985); Kasahara et al, J. Bacteriol. 173: 549-558 (1991); Su et al, Molecular
& General Genetics, 230: 28-32 (1991); Brzoska et al, "wgp-dependent transport system
for sn-glycerol 3-phosphate ofEscherichia coli" p. 170-177 in A. Torriani-Gorini, F. G.
Rothman, S. Silver, A. Wright, and E. Yagil (ed.), Phosphate Metabolism and Cellular
Regulation in Microorganisms (American Society for Microbiology, Washington, D.C.,
1987); Brzoska et al, J. Bacteriol. 176: 15-20 (1994); and Xavier etal, J. Bacteriol.
177: 699-704 (1995)).
In wild-type strains, there exists a stable intracellular pool of G3P and it is
maintained at approximately 200 uM. Internally, G3P can be synthesized by the enzymatic
conversion of glycerol by glycerol kinase (encoded by glpK) to G3P when grown on
glycerol as the sole carbon source, or from the reduction of the glycolytic intermediate,
dihydroxyactone phosphate, by G3P synthase, the gene product of the gpsA gene, during
growth on carbon sources other than glycerol. Since G3P is an important intermediate that
forms the scaffold of all phospholipid molecules, internal glycerol phosphates may also be
generated from the breakdown of phospholipids and triacylglycerol. As a metabolite,
internal G3P may be channeled into the phospholipid biosynthetic pathway or be oxidized
by G3P dehydrogenase to form dihydroxyacetone phosphate and fed into the glycolytic
pathway.
In situations where the AP promoter is employed for regulating heterologous
protein expression in E. coli, since induction occurs only after the medium is depleted of
Pi, cells induced for AP promoter activity are typically starved for phosphate and in a
declining state of health. They may have to scavenge for phosphate needed for cellular
functions. Possible consequences of such phosphate scavenging may include turnover of
ribosomes, lower cell energetics, and increased protease expression and proteolysis (St.
John and Goldberg, J. Bacteriol. 143: 1223-1233 (1980)), potentially leading to less
healthy cells with reduced capacity for protein accumulation.
Improving the metabolic state of E. coll may conceivably increase the capacity of
the cell to synthesize proteins. If phosphate is fed slowly, the cells may only sense low Pi
concentration in the periplasm, thereby inducing ihepho regulon without being starved
intracellularly for the P atom (see U.S. Pat. No. 5,304,472). There is a need for providing
further methods of producing heterologous polypeptides in E. coli.
SUMMARY OF THE INVENTION
In the invention herein, a process is provided for improving the expression of
heterologous polypeptides in E. coli. The feeding of transportable organophosphate such
as an alpha-glycerophosphate to various E. coli hosts, including those with and without
the wild-type glpT gene and those with and without the wild-type phoA gene, such as, for
example, (ugp+ kglpT phoA-) E. coli, is shown to improve the expression of
heterologous protein at both shake-flask and 10-L-fermentor scale, and is expected to
perform similarly at larger scale such as 10,OOOL. Product yield benefit was observed
across multiple model systems that employed a variety of promoters, including inducible
promoters such as the tac, T7 or AP promoter, for the expression of the heterologous
proteins. A further advantage is that the product can be obtained earlier in the active
Figure 2 shows expression of a cytoplasmic Apo2L in aHMS174 E. coli host using
the T7 promoter in a shake-flask culture, utilizing either water or 200 mM G3P as a
supplement in CRAP medium.
Figure 3 shows the effect of feeding of G3P during fermentation on secreted IGF-
1 accumulation over time. This uses a wild-type E. coli host, the AP promoter, and
continuously fed glucose.
Figure 4 shows the effect of a glpT mutation and G3P feeding during fermentation
on secreted IGF-1 accumulation over time. This uses a hglpTE. coli host, the AP
promoter, and varying G3P feed rate.
Figure 5 shows the plasmid diagram for pAPApo2-P2RU.
Figure 6 shows the nucleotide sequence of human Apo-2 ligand cDNA (SEQ ID
NO:1) and its derived amino acid sequence (SEQ ID NO:2). The "N" at nucleotide
position 447 (in SEQ ID NO:1) is used to indicate the nucleotide base may be a "T" or
Figure 7 shows the effect of G3P feeding on specific accumulation of Apo2L in
the Ag/p J E. coli (43F6) host, with three different feed rates and a control with no G3P
feed.
Figure 8 shows the benefit on the specific total accumulation of Apo2L of feeding
glycerophosphate over inorganic phosphate to the wild-type glpT host (43E7),, wherein
the cell density increases to over 200 OD550.
Figure 9 shows the effect on specific total accumulation of Apo2L of replacement
of inorganic phosphate with glycerophosphate in the wild-type glpT E. coli host (43E7)
and kglpTE. coli (43F6) host.
Figure 10 shows the effect on total Apo2L accumulation of replacement of alphaglycerophosphate
with a 50:50 mixture of alpha- and beta-glycerophosphate as a feed,
versus a no-feed control, in a AglpTE. coli (61G1) host.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
As used herein, "polypeptide" refers generally to peptides and proteins having
more than about ten amino acids. "Heterologous" polypeptides are those polypeptides
foreign to the host cell being utilized, such as a human protein produced by E. coli. While
the polypeptide may be prokaryotic or eukaryotic, preferably it is eukaryotic, more
preferably mammalian, and most preferably human.
Examples of mammalian polypeptides include molecules such as, e.g., rennin; a
growth hormone, including human growth hormone or bovine growth hormone; growthhormone
releasing factor; parathyroid hormone; thyroid-stimulating hormone;
lipoproteins; 1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; thrombopoietin;
follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors
such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting
factors such as Protein C; atrial naturietic factor; lung surfactant; a plasminogen activator,
such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin;
thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; antibodies to
ErbB2 domain(s) such as 2C4 (WO 01/00245; hybridoma ATCC HB-12697), which binds
to a region in the extracellular domain of ErbB2 (e.g., any one or more residues in the
region from about residue 22 to about residue 584 of ErbB2, inclusive); enkephalinase;
mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse
gonadotropin-associated peptide; a microbial protein, such as beta-lactamase; DNase;
inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or
growth factors; integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as
brain-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-
5, or NT-6), or a nerve growth factor such as NGF; cardiotrophins (cardiac hypertrophy
factor) such as cardiotrophin-1 (CT-1); platelet-derived growth factor (PDGF); fibroblast
growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming
growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF- 1, TGF- 2, TGF-
3, TGF- 4, or TGF- 5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(l-3)-
IGF-I (brain IGF-I); insulin-like growth factor binding proteins; CD proteins such as CD-
3, CD-4, CD-8, and CD-I9; erythropoietm; osteoinductive factors; immunotoxins; a bone
morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma;
a serum albumin, such as human serum albumin (HSA) or bovine serum albumin (BSA);
colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs),
e.g., IL-1 to IL-10; anti-HER-2 antibody; Apo2 ligand (Apo2L); superoxide dismutase; Tcell
receptors; surface-membrane proteins; decay-accelerating factor; viral antigens such
as, for example, a portion of the AIDS envelope; transport proteins; homing receptors;
addressins; regulatory proteins; antibodies; and fragments of any of the above-listed
polypeptides.
The preferred polypeptides of interest include polypeptides such as HSA, BSA,
anti-lgE, anti-CD20, anti-IgG, t-PA, gp!20, anti-CDlla, anti-CD18, 2C4, anti-VEGF,
VEGF, TGF-beta, activin, inhibin, anti-HER-2, DNase, IGF-I, IGF-II, brain IGF-I, growth
hormone, relaxin chains, growth- hormone releasing factor, insulin chains or pro-insulin,
antibodies and antibody fragments, NGF, NT-3, BDNF, Apo2L, and urokinase. The
polypeptide is most preferably IGF-1 or Apo2L.
The terms "Apo2 ligand," "Apo2L," and "TRAIL" are used herein interchangeably
to refer to a polypeptide sequence that includes amino acid residues 114-281, inclusive,
residues 95-281, inclusive, residues 92-281, inclusive, residues 91-281, inclusive, residues
41-281, inclusive, residues 15-281, inclusive, or residues 1-281, inclusive, of the amino
acid sequence shown in Figure 6 (SEQ ID NO:2), as well as biologically active fragments,
and deletional, insertional, or substitutional variants of the above sequences. In one
embodiment, the polypeptide sequence comprises residues 114-281 of Figure 6 (SEQ ID
NO:2). Optionally, the polypeptide sequence comprises residues 92-281 or residues 91-
281 of Figure 6 (SEQ ID NO:2). The Apo2L polypeptides may be encoded by the native
nucleotide sequence shown in Figure 6 (SEQ ID NO: 1). Optionally, the codon that
encodes residue Prol 19 (Figure 6; SEQ ID NO:1) may be "CCT" or "CCG." In another
preferred embodiment, the fragments or variants are biologically active and have at least
about 80% amino acid sequence identity, more preferably at least about 90% sequence
identity, and even more preferably, at least 95%, 96%, 97%, 98%, or 99% sequence
identity, with any one of the above sequences. The definition encompasses substitutional
variants of Apo2 ligand in which at least one of its native amino acids is substituted by an
alanine residue. The definition also encompasses a native-sequence Apo2 ligand isolated
from an Apo2 ligand source or prepared by recombinant or synthetic methods. The Apo2
ligand of the invention includes the polypeptides referred to as Apo2 ligand or TRAIL
disclosed in WO 97/01633, WO 97/25428, and WO 01/00832. The terms "Apo2 ligand"
and "Apo2L" are used to refer generally to forms of the Apo2 ligand that include
monomer, dimer, or trimer forms of the polypeptide. All numbering of amino acid
residues referred to in the Apo2L sequence uses the numbering according to Figure 6
(SEQ ID NO:2) unless specifically stated otherwise. For instance, "D203" or "Asp203"
refers to the aspartic acid residue at position 203 in the sequence provided in Figure 6
(SEQ ID NO:2).
The term "Apo-2 ligand extracellular domain" or "Apo2 ligand BCD" refers to a
form of Apo2 ligand that is essentially free of transmembrane and cytoplasmic domains.
Ordinarily, the BCD will have less than 1% of such transmembrane and cytoplasmic
domains, and preferably will have less than 0.5% of such domains. "Biologically active"
or "biological activity," as it relates to Apo2L, refers to (a) having the ability to induce or
stimulate apoptosis in at least one type of mammalian cancer cell or virally infected cell in
vivo or ex vivo; (b) capable of raising an antibody (i.e., immunogenic), (c) capable of
binding and/or stimulating a receptor for Apo2L; or (d) retaining the activity of a native or
naturally occurring Apo2L polypeptide.
The expression "control sequences" refers to DNA sequences necessary for the
expression of an operably linked coding sequence in a particular host organism. The
control sequences that are suitable for prokaryotes include a promoter, optionally an
operator sequence, and a ribosome-binding site.
Nucleic acid is "operably linked" when it is placed into a functional relationship
with another nucleic acid sequence. For example, DNA for a presequence or secretory
leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that
participates in the secretion of the polypeptide; a promoter is operably linked to a coding
sequence if it affects the transcription of the sequence; or a ribosome-binding site is
operably linked to a coding sequence if it is positioned so as to facilitate translation.
Generally, "operably linked" means that the DNA sequences being linked are contiguous,
and, in me case of a secretory leader, contiguous and in reading phase. Linking is
accomplished by ligation at convenient restriction sites. If such sites do not exist, the
synthetic oligonucleotide adaptors or linkers may be used in accordance with conventional
practice.
As used herein, the expressions "cell," "cell line," and "cell culture" are used
interchangeably and all such designations include progeny. Thus, the words
"transformants" and "transformed cells" include the primary subject cell and cultures
derived therefrom without regard for the number of transfers. It is also understood that all
progeny may not be precisely identical in DNA content, due to deliberate or inadvertent
mutations. Mutant progeny that have the same function or biological activity as screened
for in the originally transformed cell are included. Where distinct designations are
intended, it will be clear from the context.
The term "organophosphate" as used herein refers to a phosphate compound
containing
one or more carbon atoms, which can also contain halide atoms. Such phosphate
compound must be such that it can be fed to and utilized by a cell culture. These
compounds are often used as pesticides. "Transportable" organophosphates can be
transported from the external environment of the cell into the cell without having to be
pre-hydrolyzed in any way. If an E. coli strain does not grow well with an
organophosphate, the utilization of such organophosphate can be enhanced by
overexpressing in E. coli thsphnE gene product. Such gene confers the spontaneous
organophosphate utilization phenotype to the E. coli strain upon transformation. See
Elashvili et al, supra. Examples of suitable organophosphates include alkyl
haiophosphates such as diisopropyl fluorophosphate, alkyl phosphates such as diisopropyl
phosphate and 3,4-dihydroxybutyl-l-phosphate, as well as sugar- or alkanol-containing
phosphates such as hexose-6-phosphate and glycerol-3-phosphate. Glucose-1-phosphate,
hexose-6-phosphate and glycerophosphates such as glucose-1-glycerophosphate, fructose-
6-glycerophosphate, alpha-glycerophosphates such as glycerol-1-phosphate and glycerol-
3-phosphate, and beta-gtycero phosphate (glycerol-2-phosphate) are preferred, with
glycerophosphates more preferred, alpha- and/or beta-glycerophosphates still more
preferred, and glycerol-2-phosphate and/or glycerol-3-phosphate still more preferred, and
a mixture of glycerol-2- and glycerol-3-phosphate or glycerol-3-phosphate most
particularly preferred herein for use. As used herein, the term "G3P" without being in a
mixture or "G3P alone" refers to a composition containing at least about 80% glycerol-3-
phosphate; it may contain up to about 20% impurities such as G2P. A mixture of G3P and
G2P would contain less than about 80% G3P.
An inorganic phosphate is a phosphate compound that does not contain any carbon
atoms, with the phosphate typically being associated with an alkali or alkali earth metal
such as potassium, calcium, magnesium, or sodium phosphate.
"Active growth phase" refers to the phase of the culturing step wherein the cells are
actively growing and not severely nutrient-limited cells such as those that are in stationary
phase.
Modes for Carrying Out the Invention
The present invention provides a method for producing polypeptides heterologous
to E. coli. In this method E. coli cells comprising nucleic acid encoding the polypeptide
are cultured in a culture medium while feeding to the culture medium a transportable
organophosphate, such that the nucleic acid is expressed. The polypeptide is then
recovered from the cells. The recovery may be from the cytoplasm, periplasm, or culture
medium of the cells. The culturing may take place in any suitable vessel, preferably a
shake flask or fermentor, more preferably, in a fermentor.
Culturing parameters are used and polypeptide production may be conducted in a
conventional manner, such as those procedures described below.
A. Selection of Nucleic Acid and Modifications Thereof
The nucleic acid encoding the polypeptide of interest is suitably RNA, cDNA, or
genomic DNA from any source, provided it encodes the polypeptide(s) of interest.
Methods are well known for selecting the appropriate nucleic acid for expression of
heterologous polypeptides (including variants thereof) in E. coli.
If monoclonal antibodies are being produced, DNA encoding the monoclonal
antibodies is readily isolated and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to genes encoding the
heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred
source of such DNA. Once isolated, the DNA may be placed into expression vectors,
which are then transformed into the bacterial host cells herein to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review articles on recombinant
expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion
in Immunol. 5: 256-262 (1993) and Pliickthun, Immunol. Revs.. 130: 151-188 (1992).
Methods for humanizing non-human antibodies have been described in the art.
Preferably, a humanized antibody has one or more amino acid residues introduced into it
from a source that is non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import" variable domain.
Humanization can be essentially performed following the method of Winter and coworkers
(Jones etal, Nature, 321: 522-525 (1986); Riechmannef a/., Nature. 332: 323-
327 (1988); Verhoeyenef a/., Science. 239: 1534-1536 (1988)), by substituting
hypervariable region sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.
4,816,567) wherein substantially less than an intact human variable domain has been
substituted by the corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some hypervariable region
residues and possibly some FR residues are substituted by residues from analogous sites in
rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in making
the humanized antibodies is very important to reduce antigenicity. According to the socalled
"best-fit" method, the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain sequences. The
human sequence that is closest to that of the rodent is then accepted as the human
framework region (FR) for the humanized antibody (Sims et al., J. Immunol. 151: 2296
(1993); Chothia et al., 3. Mol. Biol. 196: 901 (1987)). Another method uses a particular
framework region derived from the consensus sequence of all human antibodies of a
particular subgroup of light or heavy chains. The same framework may be used for
several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA. 89: 4285
(1992); Prestaera/., J. Immunol. 151: 2623 (1993)).
It is further important that antibodies be humanized with retention of high affinity
for the antigen and other favorable biological properties. To achieve this goal, according
to a preferred method, humanized antibodies are prepared by a process of analysis of the
parental sequences and various conceptual humanized products using three-dimensional
models of the parental and humanized sequences. Three-dimensional immunoglobulin
models are commonly available and are familiar to those skilled in the art. Computer
programs are available that illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin sequences. Inspection of
these displays permits analysis of the likely role of the residues in the functioning of the
candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability
of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so that the desired
antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In
general, the hypervariable region residues are directly and most substantially involved in
influencing antigen binding.
Various forms of the humanized antibody or affinity-matured antibody are
contemplated. For example, the humanized antibody or affinity-matured antibody may be
an antibody fragment, such as a Fab, that is optionally conjugated with one or more
targeting agent(s) in order to generate an immunoconjugate. Alternatively, the humanized
antibody or affinity-matured antibody may be an intact antibody, such as an intact IgGl
antibody.
Fab'-SH fragments can be directly recovered from E. coli and chemically coupled
to form F(ab')2 fragments (Carter et a/., Bio/Technology. 10: 163-167 (1992)). According
to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell
culture. Other techniques for the production of antibody fragments will be apparent to the
skilled practitioner. In other embodiments, the antibody of choice is a single-chain Fv
fragment (scFv) (WO 93/16185; U.S. Pat Nos. 5,571,894 and 5,587,458). The antibody
fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641,870.
Such linear antibody fragments may be monospecific or bispecific.
Bispecific antibodies are antibodies that have binding specificities for at least two
different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of
the same protein. Bispecific antibodies can be prepared as full-length antibodies or
antibody fragments (e.g., F(ab')2 bispecific antibodies). These may be as fusions of
various antibody chains or can be one chain. One heavy chain can be competent by itself.
In one approach to producing bispecific antibodies, a bispecific immunoadhesin is
prepared by introducing into a host cell DNA sequences encoding a first fusion comprising
a first binding domain fused to an immunoglobulin heavy-chain constant domain sequence
lacking a light-chain binding site; a second fusion comprising a second binding domain
fused to an immunoglobulin heavy-chain constant domain sequence retaining a light-chain
binding site; and an immunoglobulin light-chain, respectively. The host cells are then
cultured so as to express the DNA sequences to produce a mixture of (i) a heterotrimer
comprising the first fusion covalently linked with a second fusion-immunoglobulin lightchain
pair; (ii) a heterotetramer comprising two covalently linked second fusionimmunoglobulin
light-chain pairs; and (iii) a homodimer comprising two covalently linked
molecules of the first fusion. The mixture of products is removed from the cell culture and
the heterotrimer is isolated from the other products. This approach is disclosed in WO
94/04690. For further details of generating bispecific antibodies see, for example, Suresh
era/., Methods in Enzvmologv. 121: 210 (1986).
According to another approach described in U.S. Pat. No. 5,731,168, the interface
between a pair of antibody molecules can be engineered to maximize the percentage of
heterodimers that are recovered from recombinant cell culture. The preferred interface
comprises at least a part of the Cn3 domain of an antibody constant domain. In this
method, one or more small amino acid side chains from the interface of the first antibody
molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory
"cavities" of identical or similar size to the large side chain(s) are created on the interface
of the second antibody molecule by replacing large amino acid side chains with smaller
ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For
example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other
to biotin. Such antibodies have, for example, been proposed to target immune system cells
to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of fflV infection (WO
91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made
using any convenient cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed, for example, in U.S. Pat. No. 4,676,980, along with a number
of cross-linking techniques.
Techniques for generating bispecific antibodies from antibody fragments have also
been described in the literature. For example, bispecific antibodies can be prepared using
chemical linkage. Brennan et al, Science, 229: 81 (1985) describe a procedure wherein
intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These
fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab'
fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of
the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB
derivative to form the bispecific antibody. The bispecific antibodies produced can be used
as agents for the selective immobilization of enzymes.
Additionally, Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form bispecific antibodies (Shalaby et al, J. Exp. Med.. 175: 217-
225 (1992)).
Various techniques for making and isolating bispecific antibody fragments directly
from recombinant cell culture have also been described. For example, bispecific
antibodies have been produced using leucine zippers (Kostelny et al, J. Immunol, 148.
1547-1553 (1992)). The leucine zipper peptides from the Fos and Jun proteins are linked
to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers
are reduced at the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the production of antibody
homodimers. The "diabody" technology described by Hollinger et al, Proc. Natl. Acad.
Sci. USA. 90: 6444-6448 (1993) has provided an alternative mechanism for making
bispecific antibody fragments. The fragments comprise a heavy-chain variable domain
(VH) connected to a light-chain variable domain (VL) by a linker that is too short to allow
pairing between the two domains on the same chain. Accordingly, the VH and VL domains
of one fragment are forced to pair with the complementary VL and VH domains of another
fragment, thereby forming two antigen-binding sites. Another strategy for making
bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been
reported (Gruber et al, J. Immunol. 152: 5368 (1994)).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be prepared (Tutt et al., J. Immunol.. 147: 60 (1991)).
Nucleic acid molecules encoding polypeptide variants are prepared by a variety of
methods known in the art. These methods include, but are not limited to, isolation from a
natural source (in the case of naturally occurring amino acid sequence variants) or
preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis,
or cassette mutagenesis of an earlier prepared variant or a non-variant version of the
polypeptide.
It may be desirable to modify the antibody of the invention with respect to effector
function, e.g., so as to enhance Fc receptor binding. This may be achieved by introducing
one or more amino acid substitutions into an Fc region of the antibody. Alternatively or
additionally, cysteine residues) may be introduced in the Fc region, thereby allowing
interchain disulfide bond formation in this region.
To increase the serum half-life of the antibody, one may incorporate a salvage
receptor binding epitope into the antibody (especially an antibody fragment) as described
in U.S. Pat. 5,739,277, for example. As used herein, the term "salvage receptor binding
epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgGi, IgG2, IgGs,
or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
Other modifications of the antibody are contemplated herein. For example, the
antibody may be linked to one of a variety of non-proteinaceous polymers, e.g.,
polyethylene grycol, polypropylene glycol, polyoxyalkylenes, or copolymers of
polyethylene glycol and polypropylene glycol.
B. Insertion of Nucleic Acid Into a Replicable Vector
The heterologous nucleic acid (e.g., cDNA or genomic DNA) is suitably inserted
into a replicable vector for expression in the E. coli under the control of a suitable
promoter. Many vectors are available for this purpose, and selection of the appropriate
vector will depend mainly on the size of the nucleic acid to be inserted into the vector and
the particular host cell to be transformed with the vector. Each vector contains various
components depending on the particular host cell with which it is compatible. Depending
on the particular type of host, the vector components generally include, but are not limited
to, one or more of the following: a signal sequence, an origin of replication, one or more
marker genes, a promoter, and a transcription termination sequence.
In general, plasmid vectors containing replicon and control sequences that are
derived from species compatible with the host cell are used in connection with E. coli
hosts. The vector ordinarily carries a replication site, as well as marking sequences mat
are capable of providing phenotypic selection in transformed cells. For example, E. coli is
typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g.,
Bolivar etal., Gene, 2: 95 (1977)). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides easy means for identifying transformed cells. The pBR322
plasmid, or other bacterial plasmid or phage, also generally contains, or is modified to
contain, promoters that can be used by the E. coli host for expression of the selectable
marker genes.
(i) Signal Sequence Component
The DNA encoding the polypeptide of interest herein may be expressed not only
directly, but also as a fusion with another polypeptide, preferably a signal sequence or
other polypeptide having a specific cleavage site at the N-terminus of the mature
polypeptide. In general, the signal sequence may be a component of the vector, or it may
be a part of the polypeptide-encoding DNA that is inserted into the vector. The
heterologous signal sequence selected should be one that is recognized and processed (i.e.,
cleaved by a signal peptidase) by the host cell.
For prokaiyotic host cells that do not recognize and process the native or a
eukaryotic polyp eptide signal sequence, the signal sequence is substituted by a prokaryotic
signal sequence, selected, for example, from the group consisting of the alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders.
(ii) Origin of Replication Component
Expression vectors contain a nucleic acid sequence that enables the vector to
replicate in one or more selected host cells. Such sequences are well known for a variety
of bacteria. The origin of replication from the plasmid pBR322 is suitable for most Gramnegative
bacteria such as E. coli.
(iii) Selection Gene Component
Expression vectors generally contain a selection gene, also termed a selectable
marker. This gene encodes a protein necessary for the survival or growth of transformed
host cells grown in a selective culture medium. Host cells not transformed with the vector
containing the selection gene will not survive in the culture medium. This selectable
marker is separate from the genetic markers as utilized and defined by this invention.
Typical selection genes encode proteins that (a) confer resistance to antibiotics or other
toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement
auxotrophic deficiencies other than those caused by the presence of the genetic marker(s),
or (c) supply critical nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli.
One example of a selection scheme utilizes a drug to arrest growth of a host cell.
In this case, those cells that are successfully transformed with the nucleic acid of interest
produce a polypeptide conferring drug resistance and thus survive the selection regimen.
Examples of such dominant selection use the drugs neomycin (Southern et a/., J. Molec.
Appl. Genet. I: 327 (1982)), mycophenolic acid (Mulligan et a/., Science. 209: 1422
(1980)), or hygromycin (Sugden et a/., Mol. Cell. Biol.. 5: 410-413 (1985)). The three
examples given above employ bacterial genes under eukaryotic control to convey
resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic
acid), or hygromycin, respectively.
(iv) Promoter Component
The expression vector for producing the polypeptide of interest contains a suitable
promoter that is recognized by E. coli and is operably linked to the nucleic acid encoding
the polypeptide of interest. Promoters suitable for use with E. coli hosts include the beta-
lactamase and lactose promoter systems (Chang etal. Nature, 275: 615 (1978); Goeddel
et al., Nature, 281: 544 (1979)), the arabinose promoter system (Guzman et al, I
Bacteriol.. 174: 7716-7728 (1992)), alkaline phosphatase, the T7 promoter, atryptophan
(trp) promoter system (Goeddel, Nucleic Acids Res.. 8: 4057 (1980) and EP 36,776) and
hybrid promoters such as the tac promoter (deBoer et al, Proc. Natl. Acad. Sci. USA, 80:
21-25 (1983)). However, other known bacterial promoters are suitable. Their nucleotide
sequences have been published, thereby enabling a skilled worker operably to ligate them
to DNA encoding the polypeptide of interest (Siebenlist et al., Cell. 20: 269 (1980)) using
linkers or adaptors to supply any required restriction sites.
Preferably, the promoter employed herein is an inducible promoter, i.e., one that is
activated by an inducing agent or condition (such as periplasmic phosphate depletion).
Preferred such inducible promoters herein are the alkaline phosphatase promoter, the tac
promoter, or the T7 promoter.
Promoters for use in bacterial systems also generally contain a Shine-Dalgarno
(S.D.) sequence operably linked to the DNA encoding the polypeptide of interest. The
promoter can be removed from the bacterial source DNA by restriction enzyme digestion
and inserted into the vector containing the desired DNA.
(v) Construction and Analysis of Vectors
Construction of suitable vectors containing one or more of the above-listed
components employs standard ligation techniques. Isolated plasmids or DNA fragments
are cleaved, tailored, and re-ligated in the form desired to generate the plasmids required.
For analysis to confirm correct sequences in plasmids constructed, the ligation
mixtures are used to transform E. coli K.12 strain 294 (ATCC 31,446) or other strains, and
successful transformants are selected by ampicillin or tetracycline resistance where
appropriate. Plasmids from the transformants are prepared, analyzed by restriction
endonuclease digestion, and/or sequenced by the method of Sanger et al., Proc. Natl.
Acad. Sci. USA. 7j4: 5463-5467 (1977) or Messing et al. Nucleic Acids Res.. 9: 309
(1981), or by the method of Maxam et al, Methods in Enzvmology. 65: 499 (1980).
C. Selection and Transformation of Host Cells
E. coli hosts suitable as parental hosts for expression plasmids herein include E.
coli W3110 (ATCC 27,325), E. coli 294 (ATCC 31,446), E. coli B, and E. coli X1776
(ATCC 31,537). These examples are illustrative rather than limiting. Mutant cells of any
of the above-mentioned strains may also be employed as the starting hosts that are then
further mutated to contain at least the minimum genotype required herein. E. coli
strain W3110 is a preferred parental host because it is a common host strain for
recombinant DNA product fermentations. Examples of starting E. coli hosts to be used as
parent hosts, along with their genotypes, are included in the table below:
Strain
W3110
1A2
9E4
27A7
27C6
27C7
33D3
36F8
43D3
43E7
43F6
44D6
45F8
45F9
61G1
Genotype
K-12 F lambda lN(trnD-rmE) 1
AjhuA (btonA)
kfhuA (&tonA)ptr3
A/huA (AtonA)ptr3phoA&E15 (argF-lac)169
&JhuA (ktonA) phoA&E15 (argF-lac)169 ptr3 ompT A(nmpc-fepE)
A/huA (A.tonA)phoAAE15 (argF-lac)169ptr3 degP41::kanR ompT k(nmpc-fepE)
&fhuA (htonA) ptr3 laclq lacLS ompT &(nmpc-fepE) degP::kanR
kfhuA (&tonA)phoAt\E15 (argF-lac) 1 69 ptr 3 degP41::kanR ilvG+
AJhuA (MonA) phoAAElS (argF-lac)169 ptr3 degP4 1 : :kanR ompT &(nmpc-fepE)
ilvG+
hjhuA (MonA)phoAAE15 (argF-lac) 169 ptr3 degP41 ompT &(mnpc-fepE) ilvG+
A/huA (A.tonA) phaA&ElS (argF-lac) 169 ptr3 degP41;:kanR ompT ^nmpc-fepE)
A(rbs7) ilvG+ kglpT596
AfliuA (MonA) (argF-lac) 169 ptr3 degP41::kanR ompT &(nmpc-fepE) ilvG+
AfltuA (MonA) (argF-lac) 1 69 ptr3 degP41 ompT k(nmpc-fepE) ilvG+
phoS(T10Y)
AfhuA (MonA) (argF-lac) 1 69 ptr3 degP41 ompT &(nmpc-fepE) i/vG+
phoS(T10Y) cyo::kanR
bfliuA Aptr AompTAdegP \phoA ilvG+ kglpTQ
Also suitable are the intermediates in making strain 36F8, i.e., 27B4 (U.S. Pat. No.
5,304,472) and 35E7 (a spontaneous temperature-resistant colony isolate growing better
than 27B4). An additional suitable strain is the E. coli strain having the mutant
periplasmic protease(s) disclosed in U.S. Pat. No. 4,946,783 issued August 7,1990.
In one embodiment, the E. coli host cell employed is wild type with respect to or in
reference to the g/pJgene, such as 43E7, or is deficient in the g/p^gene, such as 43F6 or
61G1. In another embodiment, the E. coli host cell employed is wild type with respect to
or in reference to ihephoA gene. In a preferred embodiment, the E. coli is deficient in
chromosomal phoA. In another preferred embodiment, the E. coli is deficient in
chromosomal glpT and in chromosomal phoA. In a more preferred embodiment, the E.
coli is deficient in chromosomal glpT and in chromosomal phoA, but not in chromosomal
ugp. The most preferred such mutant E. coli host is 43F6 or 61 Gl, the genotypes of which
are given in the above table. As used herein, "wild type with respect to glpT' refers to E.
coli hosts that areglpT+ or glpl competent cells, i.e., those that are not deficient in
chromosomal glpT. Similarly, as used herein, "wild type with respect lophoA" refers to
E. coli hosts that aiephoA+ orphoA competent cells, i.e., those that are not deficient in
chromosomal phoA.
The strains of this invention may be produced by chromosomal integration of the
parental strain or other techniques, including those set forth in the Examples below.
The nucleic acid encoding the polypeptide is inserted into the host cells.
Preferably, this is accomplished by transforming the host cells with the above-described
expression vectors and culturing in conventional nutrient media modified as appropriate
for inducing the various promoters.
Transformation means introducing DNA into an organism so that the DNA is
replicable, either as an extrachromosomal element or by chromosomal integrant.
Depending on the host cell used, transformation is done using standard techniques
appropriate to such cells. The calcium treatment employing calcium chloride, as described
in section 1.82 of Sambrook et al, Molecular Cloning: A Laboratory Manual (New York:
Cold Spring Harbor Laboratory Press, 1989), is generally used for prokaryotic cells or
other cells that contain substantial cell-wall barriers. Another method for transformation
employs polyethylene glycol/DMSO, as described in Chung and Miller, Nucleic Acids
Res.. 16: 3580 (1988). Yet another method is the use of the technique termed
electroporation.
D. Culturing the Host Cells
E. coli cells used to produce the polypeptide of interest are cultured in suitable
media as described generally in Sambrook et al, supra. The culture conditions, such as
temperature, pH, and the like, are those previously used with the host cell selected for
expression, and will be apparent to the ordinarily skilled artisan.
The cells are cultured while the culture medium is fed with a transportable
organophosphate such as a glycerophosphate, e.g., alpha-glycerophosphate and/or betaglycerophosphate,
and especially glycerol-2-phosphate and/or glycerol-3-phosphate. The
culturing may take place in a shake flask or a fermentor, preferably a fermentor. The
polypeptide is preferably recovered from the cytoplasm, periplasm, or culture medium of
the cells.
In the process of this invention, expression of the nucleic acid can begin at any
phase of the culturing step. However, preferably expression of the nucleic acid begins
while cell density is still increasing. This can be accomplished by the inducement of the
promoter with the appropriate inducer or inducing condition before cell growth ceases.
The feed rate of the organophosphate into the culture medium to be employed for
maximum production of the polypeptide depends on many factors, including the type of
organophosphate, the concentration of organophosphate, the type of polypeptide being
produced, the type of promoter, the host cell strain employed, and the cell density in the
broth. If the polypeptide is IGF-I and the organophosphate is glycerol-3-phosphate
intended to extend the production duration, under the culture conditions described and
using a 10-L process, the feed rate of the organophosphate is preferably from about 1 to 7
mmoles/hour per about 8-10 liters (see Fig. 4), more preferably from about 1 to 6
mmoles/hour, and still more preferably from about 2 to 6 mmoles/hour, yet still more
preferably from about 2 to 5 mmoles/hour, and most preferably from about 3 to 4
mmoles/hour. The optimal feed rate is dependent on the process, the cell density, the
respiration rate, etc.
Also, in a preferred embodiment, where the polypeptide is Apo2L and the
organophosphate is glycerol-3-phosphate intending to shift product expression to concur
with the active growth phase and using a 10-L process, the feed rate of the
organophosphate is from about 4 to 17 mmoles/hour per about 8-10 liters (see Fig. 7),
more preferably from about 6 to 16 mmoles/hour, still more preferably from about 8 to 15
mmoles/hour, and most preferably from about 10 to 14 mmoles/hour. The optimal feed
rate of the organophosphate needs to be determined for the individual process employed
for the expression of the specific heterologous protein.
Any other necessary media ingredients besides carbon, nitrogen, and inorganic
phosphate sources may also be included at appropriate concentrations introduced alone or
as a mixture with another ingredient or medium such as a complex nitrogen source.
Preferably, an inorganic phosphate is also present in the culture medium at the start of the
culturing step. If such inorganic phosphate, preferably sodium and/or potassium
phosphate, is present, the ratio of inorganic phosphate to organophosphate depends on
such factors as the type of polypeptide expressed and organophosphate employed. This
ratio can be any proportion, as determined readily by those skilled in the art, ranging
typically from about 1:10 (one part of Pi to 10 parts of organophosphate) to 1:0.25. For
Apo2 ligand, preferably it ranges from about 1:4 to 1:0.25, and more preferably about 1:3
to 1:0.5, and yet more preferably about 1:3 to 1:1, and still more preferably about 1:2 to
1:1, and most preferably about 1:1. Such ratios allow earlier induction of protein
expression, and in some cases allow more product to be produced earlier. The pH of the
medium may be any pH from about 5-9, depending mainly on the host organism.
If the promoter is an inducible promoter, for induction to occur, typically the cells
are cultured until a certain optical density is achieved, e.g., a Asso of about 200 for a highcell-
density process, at which point induction is initiated (e.g., by addition of an inducer,
by depletion of a medium component, etc.), to induce expression of the gene encoding the
polypeptide of interest.
Where the alkaline phosphatase promoter is employed, E. coli cells used to
produce the polypeptide of interest of this invention are cultured in suitable media in
which the alkaline phosphatase promoter can be induced as described generally, e.g., in
Sambrook et at., supra. At first, the medium may contain inorganic phosphate for the
growth of the bacterium in an amount sufficiently large to support significant cell growth
and avoid induction of synthesis of target heterologous polypeptide under the promoter
control. As the cells grow and utilize phosphate, they decrease the level of inorganic
phosphate in the medium, thereby causing induction of synthesis of the polypeptide when
the inorganic phosphate is exhausted. By adding, for example, a feed constituting a
mixture of G2P and G3P or a G3P feed, further growth to a higher cell density, such as up
to 200 OD550 or higher, takes place in the absence of inorganic phosphate or at starvation
levels of inorganic phosphate in the periplasm and supporting culture medium, resulting in
an increase or an extension of product accumulation.
E. Detecting Expression
Gene expression may be measured in a sample directly, for example, by
conventional northern blotting to quantitate the transcription of mRNA (Thomas, Proc.
Nad. Acad. Sci. USA. 77: 5201-5205 (1980)), dot blotting (RNA analysis), or in situ
hybridization, using an appropriately labeled probe, based on the sequences that encode
the polypeptide. Various labels may be employed, most commonly radioisotopes,
particularly 32P. However, other techniques may also be employed, such as using biotinmodified
nucleotides for introduction into a polynucleotide. The biotin then serves as the
site for binding to avidin or antibodies, which may be labeled with a wide variety of
labels, such as radionuclides, fluorescers, enzymes, or the like. Alternatively, assays or
gels may be employed for detection of protein.
For secretion of an expressed gene product, the host cell is cultured under
conditions sufficient for secretion of the gene product. Such conditions include, e.g.,
temperature, nutrient, and cell density conditions that permit secretion by the cell.
Moreover, such conditions are those under which the cell can perform the basic cellular
functions of transcription, translation, and passage of proteins from one cellular
compartment to another, as are known to those skilled in the art.
F. Purification of Polypeptides
The following procedures, individually or in combination, are exemplary of
suitable purification procedures, with the specific method(s) used being dependent on the
type of polypeptide: fractionation on immunoaffinity or ion-exchange columns; ethanol
precipitation; reversed-phase HPLC; hydrophobic-interaction chromatography;
chromatography on silica; chromatography on an ion-exchange resin such as SSEPHAROSE
™ and DEAE; chromatofocusing; SDS-PAGE; ammonium-sulfate
precipitation; and gel filtration using, for example, SEPHADEX™ G-75 medium. The
monoclonal antibodies may be suitably separated from the culture medium by
conventional antibody purification procedures such as, for example, protein ASEPHAROSE
™ medium, hydroxyapatite chromatography, gel electrophoresis, dialysis,
or affinity chromatography.
The invention will be more fully understood by reference to the following
examples. They should not, however, be construed as limiting the scope of the invention.
All literature and patent citations herein are incorporated by reference.
EXAMPLE 1
Feeding of G3P to Shake Flask Culture for the Production of Llama
Antibody Fragment (Heavy Chain) and Apo2L
Background:
The inclusion of 200 mM G3P (final concentration) in either low-phosphate
(CRAP) or high-phosphate culture medium (THCD) was compared to the respective
control addition (water) for the expression of a heterologous protein in shake-flask
culture. In the first part of this Example, the target heterologous protein is a 13kD llama
anti-HCG camelid monobody. Camelid antibodies have been previously shown to have 2
species, a classic IgG molecule consisting of two heavy plus two light chains and a
heavy-chain IgG molecule lacking a light chain referred to as monobody. The camelid
monobody was expressed by BL21, an E. coli B strain, using a tac promoter in either a
low-phosphate (CRAP)- or a high-phosphate (THCD) -rich media. The malE binding
protein signal sequence preceding the antibody-fragment-encoding sequence directed the
secretion of the expression protein into the periplasm of the host. In the second part of
this Example, a T7 promoter was used to regulate the expression of Apo2 ligand in
HMS174, an E. coli K12 strain, in G3P-supplemented and unsupplemented CRAP
medium. Production of heterologous protein in both experiments was induced with the
addition of IPTG upon reaching the desired cell density.
Materials & Methods:
pCB36624 86.RIG Plasmid Construction:
pCB36624_86.RIG.was constructed by modifying vector pL1602 (Sidhu et al, I
Mol. Biol. 296:487-495 (2000)). Vector pS1602, which has pTac promoter sequence and
malE secretion signal sequence, contained a sequence of human growth hormone fused to
the C-terminal domain of the gene-3 minor coat protein (p3) of phage mu. The sequence
encoding hGH was removed and the resulting vector sequence served as the vector
backbone for the insertion of a synthetic DNA fragment encoding the llama anti-HCG
antibody (Spinelli et al, Nat. Struct. Biol. 3(9): 752-757 (1996)). The resulting phagemid
(pCB36624) encoded the fusion product under the control of the IPTG-inducible Ptac
promoter (Amman and Brosius, Gene. 40:183-190 (1985)). The expressed polypeptide
included the maltose-binding protein signal peptide, followed by the anti-HCG coding
region, followed by a FLAG epitope tag, followed by a Gly/Ser-rich linker peptide
containing a suppressible stop codon, followed by P3C (the C-terminal domain of the
phage coat protein).
Phage-displayed libraries were constructed using the method of Sidhu et al, JL
Mol. Biol, 296: 487-495 (2000) with appropriately designed "stop template" phagemids.
For library NNS17, a derivative of pCB36624 that contained TAA stop codons in place of
codons 93, 94,100 and 101 was used as the template for the Kunkel mutagenesis method
(Kunkel et al, Methods Enzymol. 154: 367-382 (1987)), with mutagenic oligonucleotide
NNS17 designed to simultaneously repair the stop codons and introduce 17 NNK
degenerate codons between the codons encoding Gly95 and Trpl03.
Like all monobodies, the llama anti-HCG is a Vh3 family member and as such is
recognized by Protein A. The Protein A binding interaction was used as a surrogate for
CDR3-mediated stability. The resulting phage libraries were sorted by multiple rounds
against Protein A as readout of scaffold stability and expression. The sorted libraries were
analyzed for selection bias in the distribution of amino acids in the NNS library. Scaffold
RIG, as named by the sequence at positions 96, 97 and 98, turned out to be the most
dominant clone based on the sequenced residues. The 17-amino-acid-long CDR3 sequence
for Scaffold RIG was determined to be RIGRSVFNLRRESWVTW (SEQ ID NO:4). The
phagemid with Scaffold RIG is renamed pCB36624_86.RIG, with the DNA sequence:
petl9b.nohis Plasmid Construction
Using standard molecular biology techniques, Apo2L codons 114-281 were
amplified by polymerase chain reaction from a full-length Apo2L clone isolated from
human placental cDNA. Additional nucleotides containing restriction sites to facilitate
cloning are added to the 5' and 3' sequences, respectively. The 5' oligonucleotide primer
has the sequence:
containing the underlined Nde I restriction site. The 3' oligonucleotide primer has the
sequence:
containing the underlined BamH I restriction site. The resulting fragment was subcloned
using the restriction sites Nde I to BamH I into a modified baculovirus expression vector
pVL1392 (Pharmingen) in frame and downstream of a sequence containing a His 10 tag
and an enterokinase cleavage site (Pitti era/., J. Biol. Chem.. 271:12687-12690 (1997)).
pVL1392-Apo2L was digested with Nde I and BamH I and theMfe I-to-BamHI fragment
generated was subcloned into pET-19b (Novagen), also digested with Nde I and BamH I.
The resultant plasmid was named pet!9b.nohis.
Bacterial Strains:
Competent cells of BL21 (Stratagene) and HMS174 (Merck) were transformed
with pCB36624_86.RIG and pet!9b.nohis, respectively, using standard procedures.
Transformants were picked after growth on an LB plate containing 50ug/mL carbenicillin
(LB+CARB50™ carbenicillin), streak-purified, and grown in LB broth with 50 jag/mL
CARB50™ carbenicillin in a 30°C incubator. pCB36624_86.RIG conferred carbenicillin
resistance to the production host BL21/ pCB36624_86.RIG and petl 9b.nohis to
HMS174/petl9b.nohis, allowing the transformed hosts to grow in the presence of the
antibiotic.
Fermentation Medium:
Both low-phosphate (CRAP) culture medium and high-phosphate (THCD) culture
medium were used for the evaluation of production of llama antibody fragment and Apo2
ligand. The media composition (with the quantities of each component utilized per liter
of initial medium) is listed below:
Ingredient Low-PQ4 Medium High-PCXi Medium
Glucose
Ammonium Sulfate
Na2HPO4
NaH2P04-H20
Sodium Citrate, Dihydrate
Potassium Chloride
1 M Magnesium Sulfate
Hycase SF
Yeast Extract
Ouantitv/L
5.5 g
3.57 g
-
-
0.71 g
1.07g
7ml
5.36 g
5.36 g
Ouantitv/L
5-5 g
3.57 g
1.86g
0.93 g
0.71 g
1.07g
7ml
-
5.36 g
Casamino Acids - 5.36g
1M MOPS, 0i 7.3 110ml 110ml
KOH for pH adjustment to pH 7.3 as needed as needed
To prepare 200 mM of G3P-supplemented medium, 5 ml of 1 M DL-alphaglycerophosphate
(G3P) (Sigma Chem. Co.) was added to 20 ml of low-PC^ medium
with 50 ug/ml of carbenicillin (low-PO4 medium+CARBSO™ carbenicillin) or high-PO4
medium with 50 ug/ml of carbencillin (high-PO4 medium+CARBSO™ carbenicillin)
prior to inoculation. For the unsupplemented (control) medium, 5 ml of water was used
in place of G3P.
Shake-Flask Fermentation:
Shake-flask fermentation was conducted in a 125-ml baffled flask containing 25
ml of control or G3P-supplemented medium. An overnight culture of
BL21/pCB36624_86.RIG or HMS 174/petl9b.nohis grown in LB+CARB50™
carbenicillin was back-diluted at approx. 1:100 for inoculation into the control or G3 Psupplemented
media. Cultures were incubated at 30°C on a shaker at 250 RPM and
product expression was induced by the addition of 1 mM of IPTG when cell density
reached approximately 50-60% of the potential cell growth supported by the medium.
Cell pellets from 1 ml of broth culture, taken just before the addition of the inducer and at
approximately 24 hrs post-inoculation, were prepared and stored at -20°C.
Llama Antibody Fragment Accumulation Analyzed by PAGE and Densitometry:
Frozen (-20°C) cell pellet prepared from 1 ml of culture sample was thawed and
resuspended in sufficient quantity of lOmM TRIS, pH 7.6 + 1 mM EDTA, pH 8.0 (TE) to
bring the cell suspension to 1 OD/25 ul concentration. 25 ul of the TE-cell suspension
was mixed with 25 ul of 2X sample buffer containing beta-mercaptoethanol. The mixture
was heated at 95°C for 5 mins before 10 ul (equivalent to 0.2 OD) was loaded per well
onto NU-PAGE™ precasted 10% Bis-Tris gel (Novex). Electrophoresis was performed in
MES buffer (2-(N-morpholino) ethanesulphonic acid in deionised water adjusted to the
appropriate pH, such as with 1 N NaOH). The resolved gel was stained with
COOMASSIE BLUE R250™ stain and then destained. The band intensity of the 13-kD
antibody fragment was determined using Kodak DIGITAL SCIENCE ID™ imaging
software after scanning the wet gel with the Kodak imaging system.
Apo2 Ligand Accumulation Analyzed by Reversed-Phase HPLC:
Frozen (-20°C) cell pellet prepared from 1 ml of culture sample was resuspended
in sufficient quantity of TE buffer to bring the cell suspension to 1 OD/25 ul
concentration. 20 ul of the cell suspension was mixed into 480 ul of 6 M guanidine HC1,
pH 9.0 + lOOmM dithiothreitol (DTT), and was allowed to incubate at room temperature
for an hour before being centrifuged at 13,000 rpm for 15 mins to recover the
supernatant/extract. The extract was filtered through a MILLIPORE™ spin-filter before
20 ul was loaded onto an HPLC column (PerSeptive Biosystems POROS® Rl/10
medium) for reverse-phase chromatography. The HPLC separation was conducted at
80°C with the mobile phases flowing at 1.0 ml/min and employed a gradient of 28% to
35% of acetonitrile with 0.1% TFA over 20 minutes for the resolution of the Apo2L away
from the contaminating proteins. Peak detection was at 280 nm wavelength. The amount
of monomer present in samples was calculated using an average response factor
(mAU/ug) derived from the area under the peak associated with 5-20 ug of purified
standards analyzed by the same method.
Results:
Figure 1 shows that the antibody is expressed to higher levels in both high-PO4
(THCD) and low-PO4 (CRAP) medium supplemented with 200 mM G3P versus the
control.
Figure 2 shows that the Apo2L protein is expressed to higher levels in low-PCu
(CRAP) medium supplemented with 200 mM G3P versus the control.
EXAMPLE!
Feeding of G3P to 10-L Fermentor Culture of Wild-type or (AglpTphoA- ugp+) Host for
Production of 1GF-I Regulated by Alkaline Phosphatase Promoter
Materials & Methods:
pBKIGF-2B Plasmid for Expression of IGF-I:
The plasmid pBKIGF-2, used for the expression of IGF-I herein, was constructed
as detailed in U.S. Pat. No. 5,342,763. This plasmid was constructed from a basic
backbone of pBR322. The transcriptional and translational sequences required for
expression of the IGF-I gene in E. coli are provided by the alkaline phosphatase promoter
and the trp Shine-Dalgamo sequences. The lambda transcriptional terminator is situated
adjacent to the IGF-I termination codon. Secretion of the protein from the cytoplasm is
directed by the lamB signal sequence. The majority of rhIGF-I is found in the cell
periplasmic space. Plasmid pBKIGF-2B confers tetracycline resistance upon the
transformed host.
Bacterial Strains and Growth Conditions:
The hosts used in the IGF-I fermentation are derivatives of E. coli W3110
(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American
Society for Microbiology, 1987), pp. 1190-1219). Experiments concerning a host with
wild-typeglpTwere carried out with strain 43E7 (E. coli V*/3llQjhuA(tonA) &(argF-lac)
ptr3 degP41 &ompT&(nmpc-fepE) ilvG+ phoA), and experiments concerning a host with
a Ag/pJ mutation were carried out with strain 43F6 (E. coli W31 IQJhuAftonA) A(argFlac)
ptr3 degP41 i\ompT&(nmpc-fepE) ilvG+ phoA AglpT). Competent cells of 43E7 or
43F6 were transformed with pBKIGF-2B using standard procedures. Transformants
were picked after growth on an LB plate containing 20ng/mL tetracycline (LB +
TET20™ tetracycline), streak-purified, and grown in LB broth with 20 ^ig/mL TET20™
tetracycline in a 37°C shaker/incubator before being tested in the fermentor. pBKIGF-2B
confers tetracycline resistance to the production host and allows the transformed host to
grow in the presence of the antibiotic.
10-L Fermentation Process:
The fermentation medium composition and run protocol used for the expression
of IGF-I were somewhat similar to those used in the IGF-I process described in U.S. Pat.
No. 5,342,763. Briefly, a shake-flask seed culture of 43E7/pBKIGF-2 or 43F6/pBKIGF-
2 was used to inoculate the rich production medium. The composition of the medium
(with the quantities of each component utilized per liter of initial medium) is described
below:
Ingredient Quantity/L
Glucose200-500 g
Ammonium Sulfate 2-10 g
Sodium Phosphate, Monobasic Dihydrate 1-5 g
Potassium Phosphate, Dibasic 1 -5 g
Sodium Citrate, Dihydrate 0.5-5 g
Potassium Chloride 0.5-5 g
Magnesium Sulfate, Heptahydrate 0.5-5 g
PLURONIC™ Polyol, L61 0.1 -5 mL
Ferric Chloride, Heptahydrate 10-100 mg
Zinc Sulfate, Heptahydrate 0.1 -10 mg
Cobalt Chloride, Hexahydrate 0.1-10 mg
Sodium Molybdate, Dihydrate 0.1 -10 mg
Cupric Sulfate, Pentahydrate 0.1 -10 mg
Boric Acid 0.1-lOmg
Manganese Sulfate, Monohydrate 0.1-lOmg
Hydrochloric Acid 10-100 mg
Tetracycline 4-30 mg
Yeast Extract 5-25 g
NZ Amine AS 5-25 g
Methionine 0-5 g
Ammonium Hydroxide as required to
control pH
Sulfuric Acid as required to
control pH
A portion of the glucose, yeast extract, methionine, and NZ Amine AS is added to the
medium initially, with the remainder being fed throughout the fermentation.
The 10-liter fermentation was a fed-batch process with fermentation parameters
set as follows:
Agitation: 1000RPM
Aeration: 10.0 slpm
pH control: 7.3
Temp.: 37 °C
Back pressure: 0.3 bar
Glucose feed: computer-controlled using an algorithm to
maintain the dissolved oxygen
concentration (DC2) at 30% of air
saturation after the DO2 drops to 30%.
Complex nitrogen feed: constant feed rate of 0.2 mL/min
beginning when ODsso reaches 40 and
maintained for the remaining time of the
run
Run Duration. 40 to 50 hours
In experiments involving glycerol-3-phosphate (G3P) feeding, the appropriate
amount of 1 M G3P stock solution was spiked into the complex nitrogen feed and the
subsequent supplemented feed feed-rate increased to deliver the desired amount of
complex nitrogen plus G3P to the culture.
The impact of the &glpT mutation with or without the G3P feeding was assessed
by the difference in the IGF-I accumulation. The total amount of IGF-I in a sample
solubilized in 6M guanidine+100 mM DTT was measured by a reversed-phase HPLC
method as described in U.S. Pat. No. 6,559,122.
Results:
Figure 3 shows that with the wild-type host (43E7) and AP promoter and
continuously fed glucose, the amount of secreted IGF-I was distinctly higher when G3P
was fed to the medium than when G3P was not added.
Figure 4 shows that with the Ag/prhost (43F6) and AP promoter, the amount of
secreted IGF-I was distinctly higher when G3P was fed to the culture at 1.18 or 3.28
mmoles/hour, per approximately 8 liters, than when G3P was not added, but was not
higher when 8.22 mmoles/hour, per approximately 8 liters, of G3P was fed. The
optimum feed rate will be readily determined by one skilled in the art based on the
product, type of organophosphate, etc. Under the conditions of the fermentation process
described, culturing in a 10-liter fermentor to produce IGF-I, there is an optimal G3P feed
rate, per approximately 8-10 liters, in the preferred range of about 1-7 mmoles/hour, more
preferably about 1-6 mmoles/hour, still more preferably about 2-6 mmoles/hour, yet more
preferably about 2-5 mmoles/hour, and most preferably about 3-4 mmoles/hour. Not
only does this range of feed rates increase the amount of product over control, but also it
extends the duration of production relative to the control.
EXAMPLE 3
Feeding of Glycero-3-phosphate to Improve Apo2 Ligand Accumulation in the 10-L
Process
Background on Apo2 Ligand
Apoptosis-inducing ligand 2 (Apo2L) (Pitti et a/., J. Biol. Chem.. 271: 12687-
12690 (1996)), also known as tumor necrosis factor-related apoptosis inducing ligand
(TRAIL) (Wiley et al., Immunity. 3: 673-682 (1995)), is a type II membrane protein and
a member of the TNF family of ligands. Apo2L/TRAIL triggers apoptosis in a wide
variety of cancer cells, but not in most normal cells, through binding to its cognate death
receptors (WO 99/00423; Ashkenaa, FASEB J.. 13: (7) A1336 (April 23, 1999);
Ashkenazi, Nature Reviews - Cancer, 2: 420-430 (2002)). A soluble fragment of the
extracellular domain of Apo2 ligand, corresponding to amino acid residues 114-281
(from here on referred to as Apo2L/TRAIL), is currently under investigation for potential
clinical studies and has been successfully expressed in E. coli.
General Description of the Fermentation Process:
The expression vector encodes for the use of the alkaline phosphatase (AP)
promoter to regulate the production of the approximately 19.5-kDa polypeptide. The
expressed nascent polypeptides, upon release from the ribosomes, fold into monomers in
the cytoplasm and further associate to become the biologically active homotrimer. During
fermentation, the process parameters are set such that cellular activities are conducted at
peak oxygen uptake rates of approximately 3.0 mmoles/L-min. After broth harvest, the
cytoplasmically trapped heterologous protein is released by mechanical cell disruption
into the cell lysate from which it may be recovered.
Materials and Methods:
pAPApo2-P2RU Plasmid Construction:
pAPApo2-P2RU is described in WO 01/00832 published January 4, 2001.
Briefly, this plasmid, the construct of which is shown in Figure 5, encodes the coexpression
of Apo-2L (amino acid residues 114-281) and the rare-codon tRNA's encoded
bypro2 and argU, which co-expression is regulated by the alkaline phosphatase
promoter. The pBR322-based plasmid (Sutcliffe, Cold Spring Harbor Symp. Quant.
Biol. 43:77-90 (1978)) pAPApo2-P2RU was used to produce the Apo-2L in E. coli. The
transcriptional and translational sequences required for the expression of Apo-2L are
provided by the alkaline phosphatase promoter and the trp Shine-Dalgamo sequence, as
described for the plasmid phGHl (Chang et al, Gene, 55:189-196 (1987)). The coding
sequence for Apo-2L (from 114-281) is located downstream of the promoter and Shine-
Dalgarno sequences and is preceded by an initiation methionine. The coding sequence
includes nucleotides (shown in Figure 6) encoding residues 114-281 of Apo-2L (Figure 6
- SEQ ID NOS:1 and 2, respectively, for nucleotide and amino acid sequences) except
that the codon encoding residue Proll9 is changed to "CCG" instead of "CCT" in order
to eliminate potential secondary structure. The sequence encoding the lambda to
transcriptional terminator (Scholtissek et al, Nucleic Acids Res.. 15: 3185 (1987))
follows the Apo-2L coding sequence.
Additionally, this plasmid also includes sequences for the expression of the
tRNA's/wo2 (Komine etal, J. Mol. Biol. 212:579-598 (1990)) andargU/dnaY(Garciaet
al, Cell 45:453-459 (1986)). These genes were cloned by PCR from K coli W3110 and
placed downstream of the lambda t0 transcriptional-terminator sequence. This plasmid
confers both tetracycline and ampicillin resistance upon the production host.
Bacterial Strains and Growth Conditions:
Strain 43E7 (E. coli W3110 QiuA(tonA) phoA l\(argF-lac) ptr3 degP ompT
ilvG+)) was used as the wild-type production host for comparison to 43F6, the glpTmutated
host for the expression of Apo2 ligand and the rare codon tRNA's. Competent
cells of 43E7 or 43F6 were prepared and transformed with pAPApo2-P2RU using
standard procedures. Transformants were picked from LB plates containing 20 ug/ml
tetracycline (LB+Tet20), streak-purified, and grown in LB broth with 20 ug/ml
tetracycline in a 30°C shaker/incubator before being stored in DMSO at -80°C.
Fermentation Process for Apo2L Production:
A shake-flask inoculum was prepared by inoculating sterile LB medium containing
4-6 mM sodium phosphate with a freshly thawed stock culture vial. Appropriate
antibiotics were included in the medium to provide selective pressure to ensure retention
of the plasmid. Flask cultures were incubated with shaking at about 30°C (28°C-32°C) for
14-18 hours. This culture was then used to inoculate the production fermentation vessel.
The inoculation volume was between 0.1% and 10% of the initial volume of medium.
Production of Apo2L was carried out in the production medium given in Table 1 to
achieve a final culture volume of approximately 10 liters. The fermentation process was
conducted at about 30°C (28-32°C) and pH controlled at approximately 7.0 (6.5- 7.5).
The aeration rate and the agitation rate were set to provide adequate transfer of oxygen to
the culture. Just prior to depletion of the batched phosphate (at approximately 75-85 OD),
a DL-alpha-glycerophosphate feed (vendor product specification shows product purity at
80-90%, with beta-glycerophosphate listed as the main impurity) was initiated and fed at
the desired feed rate. Throughout the fermentation process, the cell culture was fed
glucose as the primary carbon source based on a computer algorithm while ensuring
aerobic conditions.
Two batch additions of approximately 50-150 uM (final concentration) ZnSC>4
were made during the fermentation process, one just prior to the induction of product
expression, the other at approximately the mid-point of the production period for improved
homotrimer assembly. In this example, the additions occurred at a culture optical density
of about 80-120 ODsso and at about 28 hours post-inoculation.
The fermentation was allowed to proceed for about 34-45 hours before being
harvested.
A portion of these ingredients may be fed to the culture during the fermentation.
Ammonium hydroxide was added as required to control pH.
Assessment of Soluble Product Accumulation During Fermentation Process by Ion-
Exchange HPLC Chromatography Method:
Broth samples were taken over the time course of the fermentation process. Cells
from 1 milliliter of broth samples diluted to a cell density of 20 ODsso were collected by
centrifugation and the resultant cell pellets were stored at -20°C until analysis. The cell
pellets were thawed and resuspended in 0.5 ml of extraction buffer (50mM HEPES, pH
8.0, 50 mM EDTA and 0.2 mg/ml hen egg-white lysozyme) and mechanically disrupted to
release the product from the cytoplasm. Solids were removed from the cell lysates by
centrifugation before the clarified lysates were loaded onto an HPLC column (DIONEX
PROP AC™ IEX medium) for trimer quantisation. The HPLC assay method resolved the
product away from the contaminating E. coli proteins by use of a 5%-22% gradient of 1M
NaCl in a 25-mM phosphate (pH 7.5) buffer over 25 minutes at a flow rate of 0.5 ml/min.
Assessment of Total Monomeric Apo2L Expression During Fermentation Process by
Reversed-Phase HPLC Chromatography:
Fresh culture broth or previously frozen and then thawed samples were used for
the quantitation of total monomer production. 20 ul of sample was mixed into 480 ul of
6M guanidine HC1, pH 9.0 with 100 mM DTT and was allowed to incubate at room
36
temperature for an hour before being centrifuged at 13,000 rpm for 15 mins to recover the
extract. The extract was filtered through a spin-filter before 20 ul was loaded onto an
HPLC column (PerSeptive Biosystems POROS® Rl/10 medium) for reverse-phase
chromatography. The HPLC separation was conducted at 80°C with the mobile phases
flowing at 1.0 ml/min and employed a gradient of 28% to 35% of acetonitrile with 0.1%
TFA over 20 minutes for the resolution of the Apo2L away from the contaminating
proteins. Peak detection was at 280-nm wavelength. The amount of monomer present in
samples was calculated using an average response factor (mAU/ug) derived from the area
under the peak associated with 5-20 ug of purified standards analyzed by the same
method.
Results:
Figure 7 shows an improved specific product titer (referred to as specific titer in
ug/OD-ml in the graph) with an optimum G3P feed rate to the AglpT host (43F6). All of
the G3P-fed runs performed better than the no-feed control. In this example, as the feed
rate for an approximately 8-liter culture increased from 6 to 12 mmole/hour, the specific
product titer improved, but as the rate increased above 12 mmole/hour to 18 mmole/hour,
the specific titer was lower. The optimum feed rate of G3P will be readily determined by
one skilled in the art based on the product, type of organophosphate, etc. Under these
particular conditions, culturing in a 10-liter fermentor cells for producing this specific
product, Apo2L, the preferred feed rate of G3P, per approximately 8-10 liters, is
preferably in the range of about 4 to 17 mmole/hour, more preferably about 6 to 16
mmole/hour, still more preferably about 8 to 15 mmole/hour, and most preferably about
10 to 14 mmole/hour.
Figure 8 shows an improved specific product titer (referred to as specific total
accumulation in ug/OD-ml in the graph) feeding G3P over feeding inorganic phosphate to
the wild-type glpT host (43E7). While glycerophosphate feeding increased specific total
accumulation of Apo2L, feeding inorganic phosphate negatively impacted specific total
accumulation compared to the no-feed control. Similar trends would be expected using a
lower gh/cerophosphate feed than was employed. The results here are intended to, and do,
show that a high level of expression can be obtained by feeding glycerophosphate to a
wild-type glpThost. Further, in this particular experiment, similar to the inorganic
phosphate feed case, the culture cell density increased to over 200 OD550 when the
glycerophosphate was fed, but not for the no-feed situation.
EXAMPLE 4
Expression of AP Promoter-Driven Apo2L Product during Active Growth Phase
The same plasmid construction, production host strain, medium composition,
fermentation process and product assay methods were used as described in Example 3
except for the phosphate batching and the G3P addition. A portion of the inorganic
phosphate typically included in the salt batching in a control process was replaced with an
equivalent number of moles of G3P, either added immediately after inoculation or a few
hours prior to the depletion of the batched inorganic phosphate. In these examples, the
added G3P was expected to be the source of phosphate for a significant fraction of the
cell growth subsequent to the addition.
Fermentation Process for Apo2L Production during Active Growth Phase:
The inoculum preparation protocol was the same as that described in Example 3.
Production of Apo2L was carried out in the production medium given in Table 1 except
that either 75% or 50% of the phosphate salts was eliminated from the initial batching
and replaced with an equivalent number of moles of G3P added back as a batch addition
post inoculation. The fermentation was conducted at about 30°C (28-32°C) and pH was
controlled at approximately 7.0 (6.5-7.5) as per standard protocol. The aeration rate and
the agitation rate were as described in Example 3. For the case where 50% of the
inorganic phosphate was replaced with G3P, the inorganic phosphate was batched in prior
to medium sterilization while the glycerol-3-phosphate replacement was made
approximately 1-2 hours before the batched phosphate was expected to run out (at
approximately 30-40 ODsso). For the case where 75% of the inorganic phosphate was
replaced with G3P, both the inorganic phosphate and the G3P were added immediately
after the inoculation of the fermentor. Throughout the fermentation process, the cell
culture was fed glucose as the primary carbon source based on a computer algorithm
while ensuring aerobic conditions. Zn additions were made during the fermentation
process as described in the earlier section. The fermentation was allowed to proceed for
about 34-45 hours.
Results:
Figure 9 shows the induction of heterologous protein expression occurring
significantly earlier in the active growth phase when 50% - 75% of the PO4 batching was
replaced with G3P addition for both the wild-type and g/p!T-mutated hosts, shifting the
specific total accumulation curve to the left of that for the duplicate control cases
conducted with the wild-type host with no G3P substitution. This indicates an advantage
of this invention in that the product can be obtained earlier during the fermentation
process.
While all ratios of Pi to G3P tested herein achieved this advantage regardless of
the host type, Table 2 shows that using the 1:1 or 1.3 ratio of Pi to G3P for the glpTmutated
host 43F6 produced the highest volumetric Apo2L productivity rate (an average
of about 0,34 versus an average of about 0.24 mg/ml-hr for the control host). Further,
using either ratio and the wild-type or mutated host achieved the peak specific
accumulation (in ug/OD-ml) earlier (22 to 26 hours versus 28 to 30 hours). This shows
that in certain preferred embodiments, the invention can achieve similar, if not higher,
amounts of monomeric Apo2L in approximately 10% to 25% less fermentation time than
otherwise to improve process productivity significantly.
(Table Removed) Expression of AP Promoter-Driven Apo2L Product Using a 50/50 Mixture of
Alpha- and Beta- Glycerophosphate
A procedure similar to that described in Example 3 was followed except that a
cheaper grade of approximately 50:50 mix of alpha- and beta-glycerophosphate was
employed instead of G3P as the feed using strain 61 Gl (glpT mutant host).
Results
Figure 10 shows that similar yield improvement over the no-feed control was
obtained using the mixture or the higher grade G3P material. Use of the alpha/beta
mixture would lessen the cost of raw material without compromising the production
results.

We claim:
1. A process for producing and increasing yields of a polypeptide heterologous to E. coli
comprising:
(a) culturing E. coli cells comprising a nucleic acid encoding the polypeptide in a culture
medium while feeding to the culture medium a transportable organophosphate, that is a sugar
phosphate or a glycerophosphate, such that the nucleic acid is expressed, and
(b) recovering the polypeptide from the cells, so that the yields of the polypeptide are
increased, wherein an inorganic phosphate is fed to the culture medium during the culturing
step.
2. The process as claimed in claim 1 wherein the organophosphate is a glycerophosphate.
3. The process as claimed in claim 2 wherein the glycerophosphate is an alpha-glycerophosphate or beta-glycerophosphate, or a mixture thereof.
4. The process as claimed in claim 3 wherein the glycerophosphate is a mixture of glycerol-2-phosphate and glycerol-3-phosphate or is glycerol-3-phosphate.
5. The process as claimed in claim 1 wherein the culturing takes place in a shake flask or fermentor.
6. The process as claimed in claim 1 wherein the polypeptide is recovered from the cytoplasm, periplasm or culture medium of the cells.
7. The process as claimed in claim 1 wherein expression of the nucleic acid is regulated by an inducible promoter.
8. The process as claimed in claim 7 wherein the inducible promoter is the alkaline phosphatase promoter.
9. The process as claimed in claim 7 wherein the inducible promoter is the tac promoter.
10. The process as claimed in claim 7 wherein the inducible promoter is the T7 promoter.
11. The process as claimed in claim 7 wherein expression of the nucleic acid begins while in the active growth phase of the culturing step.
12. The process as claimed in claim 1 wherein the E. coli is deficient in chromosomal phoA.
13. The process as claimed in claim 1 wherein the E. coli is wild type with respect to chromosomal glpT.
14. The process as claimed in claim 1 wherein the E. coli is deficient in chromosomal glpT.
15. The process as claimed in claim 1 wherein the E. coli is deficient in chromosomal phoA and
glpT.
16. The process as claimed in claim 15 wherein the E. coli is not deficient in chromosomal ugp.
17. The process as claimed in claim 1 wherein the polypeptide is a eukaryotic polypeptide.
18. The process as claimed in claim 1 wherein the polypeptide is a mammalian polypeptide.
19. The process as claimed in claim 1 wherein the polypeptide is insulin-like growth factor-1.
20. The process as claimed in claim 19 wherein the feed rate of the organophosphate is from about 1 to 7 mmoles/hour per about 8-10 liters and the culturing takes place in a 10-liter fermentor.
21. The process as claimed in claim 20 wherein the feed rate of the organophosphate is from about 2 to 6 mmoles/hour per about 8-10 liters.
22. The process as claimed in claim 21 wherein the feed rate of the organophosphate is from about 3 to 4 mmoles/hour per about 8-10 liters.
23. The process as claimed in claim 1 wherein the ratio of added inorganic phosphate to organophosphate ranges from about 1:10 to 1:0.25.
24. The process as claimed in claim 1 wherein the organophosphate is a hexose- 6 phosphate.
25. The process as claimed in claim 1 wherein the polypeptide is Apo2L.
26. The process as claimed in claim 25, wherein the feed rate of the organophosphate is from about 4 to 17 mmoles/hour per about 8-10 liters and the culturing takes place in a 10-liter fermentor.
27. The process as claimed in claim 26, wherein the feed rate is from about 6 to 16 mmole/hour per about 8-10 liters.
28. The process as claimed in claim 27, wherein the feed rate is from about 8 to 15 mmole/hour per about 8-10 liters.
29. The process as claimed in claim 28, wherein the feed rate is from about 10 to 14 mmole/hour per about 8-10 liters.
30. Process for producing polypeptides as claimed in any of the above claims substantially as described in the specification and illustrated in the drawings and sequence listings.